The present invention relates to a novel method for analysing nitrogen-containing compounds, for example amino acids and the like. The present invention also provides a reagent for use in the same.
Amino acids are the components from which proteins are formed, which in turn play a key role in many biological processes. In some cases the presence or absence of a particular amino acid in an individual can seriously affect their health. For example, an individual suffering from the genetic metabolic disorder phenylketonurea cannot metabolise phenylalanine; the accumulation of which severely affects their brain development. Accordingly, methods for detecting free amino acids or determining the amino acid compositions of proteins are vital for the proper diagnosis and management of diseases. Similarly, such methods are important for the analysis of commercial drugs, food and foodstuffs, as well as protein and enzyme research and development. More generally, the detection and identification of nitrogen-containing compounds finds applications across a wide range of disciplines, including, agricultural, biochemical, clinical, environmental, food, forensic, histochemical, microbiological, medical, nutritional, plant and protein sciences.
At present, free or hydrolytically released amino acids are typically detected using automatic amino acid analysers. In the 1950's the first automated amino acid analysis method was developed by Moore, Stein and Spackman, (Spackman D H, Stein W H, and Moore S. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem, 1958, 30:1190-1206). This multi-stage process involves separating the amino acids by ion exchange liquid chromatography. A ninhydrin reagent is pumped from a reagent reservoir, mixed with the eluate from the ion exchange column and passed through a steel or plastic reaction coil, and heated to the temperature required for reaction.
Ninhydrin reacts with all amino acids and related amine compounds to form highly coloured reaction products. In particular, Ruhemann's purple is formed by primary amines and primary amino acids and may be measured by absorbance of light at a wavelength of 570 nm. Yellow coloured reaction products are formed by secondary amines and a number of secondary amino acids. These reaction products may be measured by its absorbance of light at a wavelength of 440 nm. The coloured reaction products vary in intensity according to the concentration of amino acid.
The amino acid reaction products are passed through a photometer where the light absorbed by the dye compounds is detected at suitable wavelengths, in particular 570 nm and 440 nm. The presence of different amino acids may be determined by chromatography. The identity of each amino acid is established on the basis of its migration characteristics and thus its position on the chromatogram. The concentration of the amino acid is determined by the intensity of the coloured product detected in the photometer by way of absorbance at the specified wavelength. Accordingly, this method may be used qualitatively and quantitatively to determine which amino acids are present in a test sample and the relative concentrations of each.
The colour reaction between ninhydrin and the amino acid or amine is very slow at room temperature. It is significantly faster at elevated temperatures, but still takes many minutes, even at a temperature of 130° C. and above. To maintain good chromatographic performance the colour reaction needs to take place in a time period of around one minute or less. To achieve this, hydrindantin, the reduced form of ninhydrin, was found to be required for the ninhydrin reagent to be effective and provide an acceptable rate of reaction. There have been a number of suggested reasons or mechanisms for explaining the ability of hydrindantin to speed up the formation of the coloured products at elevated temperatures. One suggestion is that hydrindantin acts as a stabiliser for one of the reaction intermediates. As such it is considered as an accelerator not a catalyst.
Unfortunately, hydrindantin present in the ninhydrin reagent is a very difficult compound to handle. It is particularly unstable in the presence of air, the oxygen rapidly oxidising the hydrindantin to ninhydrin. Only relatively small amounts of air are necessary to seriously deplete the hydrindantin in the reagent and thus substantially reduce the sensitivity of the colour production in the photometric analysis. An inert atmosphere, usually nitrogen, may be used both in the preparation and during the use of the ninhydrin reagent, in an attempt to reduce the oxidation of hydrindantin. As can be appreciated, the requirement for an inert atmosphere results in the need for complex equipment and handling procedures, to ensure hydrindantin, essential for rapid amino acid analysis does not deteriorate at an unacceptable rate before and/or during its use.
In addition, hydrindantin is insoluble in totally aqueous media. Accordingly a substantial amount of organic solvent is required for storage and use of the same.
In practice, using existing equipment and techniques, it is impossible to completely exclude air during mixing or use of the ninhydrin reagent comprising hydrindantin on standard amino acid analysers. As a result, the hydrindantin concentration in the reagent will inevitably steadily decrease until no colour reaction will take place in the time frame of the chromatographic analysis. In fact, a typical ninhydrin reagent comprising hydrindantin will have a shelf life of no longer than one month and maybe as short as two weeks.
Little has changed in the last 60 years since the discovery of ninhydrin based analysis requiring hydrindantin by Moore, Stein and Spackman. Rather, this method is still the most common technique used today. However, in light of the above problems associated with using hydrindantin, slight modifications to the method have been made to minimise the rate of deterioration as much as possible. The original method by Moore. Stein and Spackman employed stannous chloride as the reducing agent. However, the amount of stannous chloride required to produce sufficient hydrindantin for a reasonably fast reaction caused eventual precipitation of tin hydroxide compounds, which in turn fouled and blocked the flow tubing. The use of stannous chloride was therefore soon abandoned and other reducing agents such as cyanide, titanous salts, borohydride and ascorbic acid were investigated. Cyanide could not be used commercially because of toxicity issues and lacked the necessary stability.
More serious studies were carried out on sodium borohydride and ascorbic acid reducing agents. For example, U.S. Pat. No. 3,778,230 and U.S. Pat. No. 4,274,833 disclose ninhydrin reagents comprising ascorbic acid and sodium borohydride or stannous chloride as reducing agents respectively. However, these reagents must first be prepared in an inert container at ambient temperatures to minimise risk of oxidation of hydrindantin. With particular reference to U.S. Pat. No. 3,778,230 and the use of ascorbic acid as reducing agent, a serious disadvantage is the production of coloured by-products which adversely affect the accuracy of the results.
Alternative techniques, some using different equipment for amino acid analysis have also been proposed. For example, U.S. Pat. No. 4,359,323 relates to a liquid chromatograph analytical system for amines. The system consists of a is chromatographic column, a reaction column and a loop to recycle the mobile phase for reuse. The primary and most significant feature of this system is that the liquid mobile phase used to separate the amino acid sample is comprised of a combination of an eluent and a substance which reacts with amines to produce a compound which can be detected photometrically. Accordingly, this process only requires a single pump for complete operation. In this way, exposure of hydrindantin to the surrounding air is supposedly reduced. However, there is still a significant risk with this invention that the reactant will interfere with the amino acid sample within the separation column.
Unlike the Moore. Stein and Spackman method, Hitachi is understood to mix sodium borohydride and ninhydrin together on-line before the heated reaction chamber. Accordingly, it has been claimed that the Hitachi reagent may be used for up to a maximum of 12 months, rather than the standard 1 month of a mixed reagent. Nevertheless, sodium borohydride is still not fully stable and it is known that trace amounts of metals can accelerate its deterioration. As a result, a significant amount of the sodium borohydride could be lost before the end of one year. Although hydrindantin is produced on-line, an extra pumping system or line is necessary for inserting sodium borohydride into the analysis equipment. This increases the complexity and cost of the system and the Hitachi reagent will not be usable on other manufacturers' instruments, unless they are fitted with an additional pumping system and supply lines.
An alternative technique for preparing a ninhydrin reagent is disclosed in U.S. Pat. No. 3,632,496. A reagent generator is formed from an elongated housing having a channel therethrough including an inlet at one end for receiving a reagent and an outlet at the other for discharging activated reagent. The channel is defined by surfaces of metallic material catalytically active for reducing ninhydrin to form the activated reagent. In this way, ninhydrin in its stable state may be stored and activated in one vesicle. However, this invention provides an overly complicated mechanism and device for preparing activated ninhydrin reagents. As discussed above, manufacturers have since reverted to using a much simpler method, wherein ninhydrin and hydrindantin, stored separately, are mixed together immediately prior to use on the amino acid analyser. Furthermore, the reagent reactor of U.S. Pat. No. 3,632,496 is to be incorporated within a reagent sprayer, for use in applications of the field of paper chromatography. Accordingly, transferral of the activated reagent from the reagent reactor into the amino acid analyser equipment for use is likely to be difficult, if not impossible without exposing the hydrindantin to the surrounding air.
Whilst there has been considerable research into finding alternative ninhydrin reagents, in particular, alternative reducing agents, there is still a need for an improved method, providing an effective, simple and inexpensive way of analysing amino acids and the like. It would be particularly advantageous if an alternative method was available for use with a wide range of ninhydrin reagents.